초록

○ 연구결과
1. Resveratrol 고생산 유전자 발현 시스템 개발 및 함량 증진 환경 시그널 탐색 연구
2. 포도 형질전환 기술 개발 및 resveratrol 고생산 포도 형질전환체 개발

Abstract ▼

IV. Results and Further Application
1. Results
1) Development of a transgenic vector system for grapevine and a probing system for environmental signals increasing the content of resveratrol in domestic grapevine cultivars.
A stilbene synthase cDNA clone was isolated from grapevine Kyoho an

IV. Results and Further Application
1. Results
1) Development of a transgenic vector system for grapevine and a probing system for environmental signals increasing the content of resveratrol in domestic grapevine cultivars.
A stilbene synthase cDNA clone was isolated from grapevine Kyoho and the open reading frame of the clone was fused with CaMV 35S promoter to construct a transgenic vector system and incorporated into tobacco. Resveratrol production(0.45㎍ /g) was confirmed in leaves of transgenic plants by HPLC. This result suggested that the incorporation of the cDNA clone was sufficient to produce resveratrol.
The stsy gene expression profile was analysed in different organs at the various stages of development with or wothout environmental signals such as UV, AlCl₃, elicitor(yeast extract). UV treatment was the most efficient to induce the stsy gene expression. However, a more favorable condition which could be applied in the field was required and a probing system was therefore developed to search for environmental signals inducing the resveratrol biosynthesis. Four different homologous stilbene synthase genes including the promoter regions were cloned by PCR-based DNA walking from grapevine Kyoho and their promoter regions were very homologous among them while the open reading frames were identical in the nucleotide suquence. The stsy promoter 1, the longest one, contained various cis-elements such as GATA motif, GCC-box, CAAT, HES(heat response element), WUN(wound response element), G box, GT 1, AS-1, and other light-responsive elements I box, AE box and GAG motif.
The stsy promoter 1 was fused to the GUS reporter gene and incorporated into tobacco and Arabidopsis. Yeast extract, UV and AlCl₃ seemed to induce the resveratrol biosynthesis in leaves, roots and stems of transgenic plants. These results suggest that the stsy promoter region may be applied to construct a probing system for environmental signals increasing the content of resveratrol in domestic grapevine cultivars. In addition, identification and characterization of the cis-elements involved in the environmental stress response will result in the development of cultivation techniques to improve the resveratrol content. Deletion series of the promoter 1 was constructed and incorporated into Arabidopsis to search for the environmental signals which were able to applied to vineyard fields to increase the resveratrol content. The selected signals were applied to the grapevine cuttings and confirmed to induce the expression of the stsy transcripts. These elements may be used to construct an inducible and strong gene expression system.
2) Development of a transformation system and transgenic grapevine plants with high contents of resveratrol in berries
A plant regeneration system via organogenesis and Agrobacterium tumefaciens mediated transformation was established for grapevine (Vitis sp.). The effects of medium composition on shoot organogenesis from the leaf segments of in vitro shoot cultures were tested. Among the plant growth regulators, the highest regeneration rate was obtained in MS medium with BA 1.0 mgㆍL^-1 and IBA 0.05 mgㆍL^-1 (46.7%). TDZ 0.5 mgㆍL^-1 and IBA 0.05 mgㆍL^-1 showed relatively high regeneration rate (26.7%) and less browning. Among gelling agents, agarose produced the highest regeneration rate (70.0%) but caused browning. Agar of 6 gㆍL^-1 showed high regeneration frequency and little browning. This regeneration system produced shoot regeneration frequency of 83.3% and 1.5 shoots per explant.
For the development of a transformation system, leaf segment explants were transformed with EHA105/pCAMBIA2301 and the transformation efficiency was tested by histochemical GUS analysis. The optimal conditions for transformation were as follows: pre-culture 1 day, inoculation time 10 min, inoculation 0.5(OD600nm), and co-cultivation 2 days. Four genotypes of grapes, 'Schuyler', 'Tamnara', 'Kyoho' and SO4, were transformed using Agrobacterium LBA4404/pCAMBIA2301. Only 'Schuyler' produced shoots from the transformed explants after 2 months in culture. The regenerated shoots were successfully multiplied and rooted. Gene expression of the transformed shoots was confirmed through PCR and GUS analysis. We developed an Agrobacterium-mediated transformation system and produced transgenic plants via indirect shoot organogenesis in grapevine. To produce transgenic plants with a high content of resveratrol, 'Schulyer' was transformed with Agrobacterium LBA4404/pBIstsy. Calli were obtained but shoot was not produced from transformed explants yet. However, with the transformed calli, incorporation and expression of the Kyoho stilbene synthase gene were confirmed with genomic PCR and RT-PCR analyses. The stilbene synthase gene expresion was also detected by measuring the resveratrol content in transgenic calli by HPLC. These results suggest that the developed transformation system has been well established for grapevine.
3) Cultivation of Transgenic Grapevine Plants and Development of Cultivation Techniques for Improving the Resveratrol Content Using Environmental Signals
This study was conducted to survey the varietal and regional differences in resveratrol content, and to examine the seasonal changes and the distribution pattern of resveratrol in ‘Campbell Early’ grapes (Vitis spp.). Some elicitors along with yeast and one commercial bacterial formula were also tested for their effects on resveratrol content and fruit quality.
Resveratrol content in fruit skin was in the order of ‘Delaware(2.63 μg/g)’, ‘Campbell Early(2.58 μg/g)’, and ‘Kyoho(2.42 μg/g)’ while that of ‘Shigyoku’ and ‘Red Globe’ was low. Pericarp of ‘Campbell Early’ was composed of one layer of epidermis and 7 to 8 layers of hypodermis, while that of ‘Kyoho’ was composed of one layer of epidermis and 3 to 4 layers of hypodermis. And the differences in number of cell layers were not related to resveratrol content. Resveratrol content in berry skin and seed was 2.58 μg/g and 0.50 μg/g respectively, while flesh contained no resveratrol.
Resveratrol was detected in leaves and berries throughout growing season with temporary decrease around veraison, and the onset of production was not clear.
Resveratrol content of ‘Campbell Early’ grape was higher at harvest than veraison regardless of area. And resveratrol content of grapes grown in Yanggu area was remarkably higher than those grown in Suwon and Daebudo area at both veraison and harvest with respective 0.69 μg/g and 3.90 μg/g.
In non-bagged culture, number of cell layers constituting pericarp at veraison decreased by more than half at harvest in grapes grown in Suwon and Daebudo area, while those grown in Yanggu area showed a slight decrease from 9∼13 layers to 8∼10 layers.
2. Further Application
According to the significance as the above, this study aimed at the development of the transgenic grapevine with a high content of resveratrol by means of reprogramming biosynthetic genes of resveratrol through the genetic engineering techniques, and the development of cultivation techniques to increase resveratrol contents in fruits of the major domestic grapevine cultivars using environmental signals. Particularly this study will induce increase of grape consumption and export through development and cultivation of highly qualified functional grapes under the present circumstances that the agricultural basis in the country continuously becomes weakened, and intends to promote self-admiration of the agriculture.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 9
• CONTENTS ... 14
• 목차 ... 16
• 제 1 장 연구개발과제의 개요 ... 18
• 제 1 절 연구개발의 필요성 ... 18
• 제 2 절 연구개발의 목표 및 내용 ... 23
• 제 2 장 국내외 기술 개발 현황 ... 24
• 제 1 절 국외 관련기술의 현황 ... 24
• 제 2 절 국내 관련기술의 현황 ... 26
• 제 3 절 앞으로의 전망 ... 26
• 제 3 장 Resveratrol 생합성 유전자 형질전환용 vector 및 발현 증진 환경 시그널 탐색 시스템 개발 ... 27
• 제 1 절 stilbene synthase genomic clone 및 promoter확보 ... 27
• 제 2 절 형질전환용 벡터 및 환경인자 탐색 시스템 개발 ... 42
• 제 3 절 promoter deletion series 분석을 통한 환경 시그널 탐색 ... 50
• 제 4 장 Resveratrol 함량증진 환경 시그널 탐색 ... 58
• 제 1 절 서론 ... 58
• 제 2 절 연구 방법 ... 58
• 제 3 절 연구 결과 및 고찰 ... 60
• 제 4 절 참고 문헌 ... 64
• 제 5 장 포도형질전환 기술개발 및 resveratrol 고생산 포도 형질전환체 개발 ... 68
• 제 1 절 서론 ... 68
• 제 2 절 실험재료 및 방법 ... 70
• 제 3 절 연구결과 및 고찰 ... 78
• 제 4 절 적요 ... 100
• 제 5 절 참고문헌 ... 101
• 제 6 장 형질전환 포도 신품종 재배 및 환경 시그널을 이용한 resveratrol 함량 증진 포도재배 기술개발 ... 106
• 제 1 절 실험재료 및 방법 ... 106
• 제 2 절 연구결과 및 고찰 ... 113
• 제 3 절 참고문헌 ... 137
• 제 7 장 목표달성도 및 관련분야에의 기여도 ... 141
• 제 8 장 연구개발결과의 활용계획 ... 144
• 제 9 장 연구개발과정에서 수집한 해외과학기술정보 ... 145
• 끝페이지 ... 146